An optimization method for preparing amniotic membrane stem cell membrane
By adding the NAD+ precursor compound NMN to the vitamin C-induced culture medium and combining it with mechanical scraping, the problems of long preparation cycle and high proportion of CD34 positive cells in amniotic stem cell membrane preparation were solved, achieving efficient and low immunogenic membrane preparation.
Patent Information
- Authority / Receiving Office
- CN · China
- Patent Type
- Applications(China)
- Current Assignee / Owner
- ZHEJIANG HANGKE CELL ENG CO LTD
- Filing Date
- 2026-01-29
- Publication Date
- 2026-05-29
AI Technical Summary
Traditional amniotic stem cell membrane preparation has a long cycle and a high proportion of CD34-positive cells, which poses a risk of immune rejection.
The NAD+ precursor compound NMN was added to the vitamin C-induced culture medium, and cell membrane sheets were separated by mechanical scraping.
It significantly shortens the preparation cycle to 7-10 days, reduces the proportion of CD34 positive cells to below 0.12%, improves membrane purity and immunogenicity, and enhances the structure of the extracellular matrix.
Smart Images
Figure CN122104568A_ABST
Abstract
Description
Technical Field
[0001] This invention relates to the fields of tissue engineering and regenerative medicine, specifically a method for preparing amniotic stem cell membranes based on NAD+ precursor compounds, and the application of these membranes, particularly suitable for the preparation of low-immunogenic tissue repair materials. Background Technology
[0002] Amniotic stem cells (hAMSCs) are considered ideal seed cells for tissue engineering due to their wide availability, strong proliferative capacity, low immunogenicity, and multi-lineage differentiation potential. Cell sheet technology is a method that can obtain complete cell layers without enzymatic digestion, effectively preserving the extracellular matrix and intercellular connections, and has shown great potential in areas such as myocardial repair, skin regeneration, and osteochondral reconstruction.
[0003] Traditional methods for preparing cell sheets often involve adding high doses of vitamin C to promote the synthesis and secretion of the extracellular matrix (mainly collagen), thereby encouraging cells to form intact membranes during detachment. However, this method has two significant drawbacks: (1) The preparation cycle is long, usually requiring 2-3 weeks; (2) The obtained cell sheet often contains a small number of CD34-positive epithelial cells or crude cells, and their expression level is usually above 2%. As a marker of hematopoietic stem / progenitor cells, the presence of CD34 may indicate the heterogeneity of the cell population and potentially increase the risk of immune rejection after transplantation.
[0004] Therefore, how to shorten the preparation time and further reduce the CD34 positivity rate without affecting the membrane formation ability and stem cell function is a key issue in improving the clinical applicability of amniotic stem cell membranes. Summary of the Invention
[0005] This invention provides an optimized method for preparing amniotic stem cell membranes to solve the above-mentioned problems.
[0006] To achieve the above objectives, the present invention provides the following technical solution: An optimized method for preparing amniotic stem cell membrane sheets includes the following steps: (1) Amniotic stem cells were seeded into a culture dish and cultured. (2) When the cells adhere to the wall and grow to a degree of confluence of more than 80% and the cells are in good condition, replace the medium with an induction medium containing vitamin C (VC); during the second medium change, add an NAD+ precursor compound to the induction medium; the NAD+ precursor compound is β-nicotinamide mononucleotide (NMN) or nicotinamide adenine dinucleotide (NAD+). (3) Continue culturing for 7-10 days, changing the induction medium regularly during this period, until a complete cell membrane is formed; (4) The cell membrane sheet is mechanically peeled off from the bottom of the culture dish to obtain an intact amniotic stem cell membrane sheet.
[0007] Preferably, the specific method and concentration of vitamin C addition are as follows: The concentration of vitamin C (VC) in the culture medium is 10-100 μg / mL, preferably 20 μg / mL; Vitamin C (VC) was first added when more than 80% of the amniotic stem cells adhered to the culture medium and the cells were in good condition (cell viability ≥95%, spindle-shaped or polygonal shape and uniform adhesion, no obvious apoptotic bodies, and a 24-hour proliferation rate ≥1.5). The culture medium containing the same concentration of vitamin C (VC) was changed every two days.
[0008] Preferably, the specific method and concentration of the NAD+ precursor compound are as follows: The concentration of NMN in the culture medium is 0.01-0.1 mM, and the concentration of NAD+ is 0.1-1 mM, preferably 0.1 mM for NMN and 1 mM for NAD+. The NAD+ precursor compound was added along with vitamin C (VC) during the second medium change, and the induction medium containing vitamin C (VC) and the NAD+ precursor compound was changed every two days thereafter.
[0009] Preferably, in step (4), the mechanical dissection is performed using a sterile cell scraper or forceps; specifically: after the amniotic stem cell membrane has been cultured for 7-10 days, when the cells are observed to be significantly thickened and a curled white film appears at the edge of the culture dish, the cell membrane is carefully separated from the bottom of the cell culture dish using a sterile cell scraper, and the separated amniotic stem cell membrane is intact and without wrinkles; specifically: the scraper is at an angle of 15-30° to the culture dish, and a circle is first gently scraped around the edge of the culture dish to initially separate the edge of the membrane, and then scraped unidirectionally from the edge to the center, avoiding pressing or scraping back and forth; the separated membrane can be transferred to sterile PBS buffer for temporary storage (stored at 4°C for no more than 24 hours) to obtain an intact amniotic stem cell membrane without wrinkles.
[0010] This invention involves adding NAD+ or its key precursor NMN to a conventional induction culture medium containing VC, and then using a mechanical scraping method to separate intact, non-wrinkled amniotic stem cell membranes.
[0011] NAD+ is a core coenzyme for cellular energy metabolism and signal transduction, participating in the regulation of various life activities such as cell proliferation, apoptosis, aging, and oxidative stress. Supplementing with NMN / NAD+ has the following effects: (1) Enhances cellular energy metabolism: Significantly increases intracellular NAD+ levels, accelerates ATP production, provides sufficient energy for the high-energy-consuming matrix synthesis process of cells, thereby greatly shortening membrane formation time.
[0012] (2) Regulation of cell fate: By activating the longevity protein family such as Sirtuins, the differentiation state and surface marker expression of stem cells are regulated; the present invention unexpectedly found that the addition of NMN / NAD+ can specifically inhibit the expression of CD34, promote the enrichment of amniotic stem cells into a more purified mesenchymal stem cell population, and reduce immunogenicity.
[0013] The present invention also provides an amniotic stem cell membrane, which is prepared by the above method. The percentage of CD34 positive cells in the membrane is less than 0.12%, preferably less than 0.09%, and the membrane has an intact cell-matrix structure.
[0014] Observation using a scanning electron microscope (SEM) revealed that the cells were arranged in a tight and orderly manner, and the cell nuclei were clearly visible, with a significantly increased cell density.
[0015] Protein analysis by LC-MS revealed that the membrane contained a high content of type VI collagen, which enhanced the membrane's mechanical integrity, elasticity, and toughness, resulting in a superior membrane structure.
[0016] The present invention also provides the use of the above-mentioned amniotic stem cell membrane in the preparation of drugs or medical devices for treating corneal damage, skin trauma, knee joint filling or endometrial adhesions.
[0017] Compared with the prior art, the beneficial effects of the present invention are as follows: 1. Significantly shorten the preparation cycle: Reduce the traditional 14-21 days to 7-10 days, improving preparation efficiency and reducing the risk of contamination.
[0018] 2. Significantly reduced CD34 expression: The proportion of CD34-positive cells in the membrane was successfully reduced from about 2% to below 0.12%, resulting in stem cell membranes with higher purity and lower immunogenicity.
[0019] 3. Simple operation and easy standardization: This method is an optimization of existing mature technology. It only requires the addition of specific components to the culture medium and simple mechanical operation, which makes it easy to promote and apply in routine cell laboratories.
[0020] 4. Excellent membrane quality: The prepared membrane has high cell viability, abundant extracellular matrix, intact structure, high content of type VI collagen, and good mechanical properties, making it more suitable for clinical transplantation. Attached Figure Description
[0021] Figure 1This is a bar chart showing the unit cell density after each membrane was formed in Example 2.
[0022] Figure 2 This is a flow cytometry result of the CD34 expression rate in each membrane in Example 3.
[0023] Figure 3 This is a protein composition analysis diagram of the optimized amniotic stem cell membrane in Example 4.
[0024] Figure 4 This is an electron micrograph of the amniotic stem cell membrane sheet cells optimized in Example 5.
[0025] Figure 5 This is an analysis diagram of the elastic modulus of the amniotic stem cell membrane optimized in Example 6.
[0026] Figure 6 This is a flowchart of the optimized method for preparing amniotic stem cell membranes according to the present invention. Detailed Implementation
[0027] Unless otherwise defined, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this application belongs; the terminology used herein in the specification of the application is for the purpose of describing particular embodiments only and is not intended to be limiting of the application; the terms "comprising" and "having," and any variations thereof, in the specification, claims, and foregoing drawings of this application are intended to cover non-exclusive inclusion. The terms "first," "second," etc., in the specification, claims, or foregoing drawings of this application are used to distinguish different objects, not to describe a particular order.
[0028] In this document, the term "embodiment" means that a particular feature, structure, or characteristic described in connection with an embodiment may be included in at least one embodiment of this application. The appearance of this phrase in various places throughout the specification does not necessarily refer to the same embodiment, nor is it a separate or alternative embodiment mutually exclusive with other embodiments. It will be explicitly and implicitly understood by those skilled in the art that the embodiments described herein can be combined with other embodiments.
[0029] Example 1 This invention provides an optimized method for preparing amniotic stem cell membrane sheets, such as... Figure 6 As shown, it includes the following steps: 1. Cell inoculation Amniotic stem cells (P3-P5 generation, derived from full-term healthy placental amnion) in good growth condition and in the logarithmic growth phase were collected. After digestion with a mild digestive enzyme (using a mixture of 0.25% trypsin and 0.02% EDTA at 37°C for 5-8 minutes, followed by centrifugation to collect the cells), they were distributed at a concentration of 1 × 10⁻⁶ cells per square centimeter. 5Cells were evenly seeded at a density of 1,000 cells per well in 6-well cell culture plates. They were pre-cultured for 24 hours in amniotic stem cell culture medium (DMEM / F12 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin (100 U / mL penicillin, 100 μg / mL streptomycin) to ensure that the cells adhered fully to the plate and covered more than 80% of the bottom of the culture dish.
[0030] 2. Optimization of amniotic stem cell sheet culture preparation a. When the cells have covered more than 80% of the bottom of the culture dish and are in good condition (cell viability ≥95%, spindle-shaped or polygonal shape and uniform adhesion, no obvious apoptotic bodies, and a 24-hour proliferation rate ≥1.5), discard the old culture medium; use a pipette to slowly and gently add 10 ml of induction medium containing 20 μg / ml vitamin C along the edge of the culture dish. b. Repeat the above steps, changing the culture medium every two days. During the second culture medium change, add NAD+ precursor compounds (0.1 mM NMN and 1 mM NAD+) and vitamin C at 20 μg / ml. Then change the induction culture medium containing vitamin C and NAD+ precursor compounds every two days thereafter. c. Observe the morphology of the cell membrane for 7-10 days. When the cells in the culture medium thicken significantly and the edges of the cell culture dish curl and a white film appear, carefully separate the cell membrane from the bottom of the cell culture dish using autoclaved forceps or a cell scraper. The separation parameters are: the angle between the scraper and the culture dish is 15-30°. First, gently scrape around the circumference of the culture dish to initially separate the membrane edge, and then scrape unidirectionally from the edge to the center, avoiding pressing or scraping back and forth, to obtain an intact amniotic stem cell membrane without wrinkles. Comparative Example 1: Traditional VC Induction Method (VC Only) This comparative example provides a method for preparing amniotic stem cell sheets based on the traditional VC induction method, including the following steps: 1. Cell inoculation Amniotic stem cells (P3-P5 generation, derived from full-term healthy placental amnion) in good growth condition and in the logarithmic growth phase were collected, digested with a mild digestive enzyme, and then added at a concentration of 1×10⁻⁶ cells / cm². 5 Cells were evenly seeded at a density of 1,000 cells per well in a 6-well cell culture plate and pre-cultured for 24 hours in amniotic stem cell culture medium to ensure that the cells adhered fully to the plate and covered more than 80% of the bottom area of the culture dish.
[0031] 2. Preparation of amniotic stem cell membrane sheets a. When the cells have covered more than 80% of the bottom of the culture dish and are in good condition, remove and discard the old culture medium; use a pipette to slowly and gently add 10 ml of induction medium containing 20 μg / ml vitamin C along the edge of the culture dish. b. Repeat the above steps, changing the culture medium every two days for 14-21 days. Observe the cell membrane morphology. When the cells in the culture medium are significantly thickened and the edges of the cell culture dish are curled and a white film appears, carefully separate the cell membrane from the bottom of the cell culture dish using autoclaved forceps or a cell scraper.
[0032] This comparative example is used to demonstrate the key role of NAD+ precursor compounds. Cells need to be cultured for 17-21 days to form structurally intact, retrievable membranes.
[0033] Comparative Example 2: No VC induction (basal medium) This comparative example was used to demonstrate the fundamental role of VC in extracellular matrix secretion. The procedure was exactly the same as that of Comparative Example 1, but neither VC nor NMN / NAD+ was added to the induction medium. The cells were cultured in DMEM / F12 medium containing 10% fetal bovine serum (FBS) and 1% penicillin-streptomycin. The cells could only proliferate to the point of fusion, but could not secrete enough extracellular matrix to form a peelable membrane.
[0034] Example 2 To objectively evaluate the effectiveness of the present invention, a series of tests were performed on the membranes prepared in the above embodiments and comparative examples. The membrane formation time and morphological observation results are shown in Table 1 below: Table 1. Membrane formation time and morphological observation. Cell density of each membrane patch as follows Figure 1 As shown above, the method of the present invention (Example 1) significantly shortens the membrane preparation cycle (increasing efficiency by about 50%), improves the physical properties of the membrane, and increases the unit cell density of the membrane.
[0035] Example 3 Flow cytometry was used to detect CD34 expression rate, such as Figure 2 As shown; the collected membranes were digested into single-cell suspensions using type IV collagenase, stained with anti-human CD34-APC antibody, and analyzed by flow cytometry: Comparative Example 1 (VC only): CD34 positive cell rate was 2.04%; Example 1 (VC+NMN group): CD34 positive cell rate was 0.12%; Example 1 (VC+NAD+ group): CD34 positive cell rate was 0.09%.
[0036] The method of this invention can significantly reduce the proportion of CD34-positive cells in the membrane, demonstrating the unique role of NMN / NAD+ in regulating stem cell surface markers and reducing immunogenicity.
[0037] Example 4 Optimized analysis of amniotic stem cell membrane protein components, such as Figure 3 As shown; the key protein composition of the amniotic stem cell membrane prepared in this invention was analyzed by liquid chromatography-mass spectrometry (LC-MS) to confirm its superior matrix composition. Specific implementation methods are as follows: 1. Membrane source: amniotic stem cell membrane prepared according to the method in Example 1.
[0038] 2. Protein dissolution and enzymatic digestion: The decellularized ECM matrix was dissolved in 8 M urea solution; it was reduced with dithiothreitol (DTT) and alkylated with iodoacetamide (IAA); then it was enzymatically digested overnight at 37°C using sequencing-grade trypsin.
[0039] 3. Peptide desalting: The peptide mixture after enzymatic hydrolysis is desalted and purified by passing it through a C18 desalting column. After vacuum centrifugation and drying, it is reconstituted with 0.1% formic acid solution for instrumental detection.
[0040] 4. LC-MS detection steps: a. Liquid Chromatography (LC) Separation: A nano-flow LC system was used; peptide samples were first loaded onto a C18 pre-column for enrichment and desalting, and then separated onto an analytical column (column temperature 50°C); the following gradient was used for separation: mobile phase A was 0.1% formic acid aqueous solution, and mobile phase B was 0.1% formic acid acetonitrile solution; the gradient started at 5% B, linearly increased to 28% B within 90 minutes, and then increased to 95% B within 5 minutes and maintained for 5 minutes to clean the column.
[0041] b. Mass spectrometry (MS) detection: The separated peptides are detected by a tandem mass spectrometer; the mass spectrometer is operated in data-dependent acquisition (DDA) mode; the full scan resolution is 70,000, and the scan range (m / z) is 350-1200; after each full scan, the top 20 precursor ions with the highest intensity are selected for high-energy collisional fragmentation (HCD), and the fragmentation normalization energy is set to 28%.
[0042] 5. Data Analysis: a. Import the obtained raw mass spectrometry data into proteomics analysis software (such as Proteome Discoverer 2.4); use a search engine (such as Sequest HT) to search the human protein database (UniProtKB), paying particular attention to entries for extracellular matrix proteins.
[0043] b. Set search parameters: trypsin digestion, allowing a maximum of 2 missed cleavage sites; precursor ion mass tolerance is 10 ppm, fragment ion mass tolerance is 0.02 Da; fixed modification is cysteine alkylation, variable modification is methionine oxidation.
[0044] c. Screen the identified proteins (false positive rate FDR < 1%) and perform relative quantitative analysis based on mass spectrometry counts and precursor ion area.
[0045] 6. Results: LC-MS analysis successfully identified hundreds of proteins, with the following key proteins showing significant relative enrichment (p<0.05): Type VI collagen forms a unique microfibril network that connects cells to the macromatrix and plays a central role in regulating cell behavior. Type I collagen is a major structural component of the ECM, providing strong mechanical support; Fibrillin-1, a major component of microfibrils, is essential for maintaining tissue elasticity and stability.
[0046] This LC-MS proteomics analysis example demonstrates at the molecular level that the amniotic stem cell membrane prepared by the method of this invention (with the addition of NAD+ precursor) exhibits significant enrichment of type I collagen, type VI collagen, and fibrinogen, which together constitute a cellular microenvironment that is closer to natural tissue and has excellent mechanical properties and biological functions. This allows the membrane to exhibit better tissue integration and repair capabilities after transplantation.
[0047] Example 5 Optimized scanning electron microscopy (SEM) ultrastructural analysis of amniotic stem cell membrane sheets, such as Figure 4 As shown, the specific implementation method is as follows: 1. Sample preparation: a. Fixation: Take the amniotic stem cell membrane prepared according to the method of Example 1; gently rinse the collected intact membrane three times with sterile PBS to remove culture medium residue; then immediately perform primary fixation with 2.5% glutaraldehyde (in 0.1 M phosphate buffer, pH 7.4) at 4°C for a soaking time of not less than 4 hours; after fixation, rinse again with 0.1 M phosphate buffer three times, 15 minutes each time.
[0048] b. Post-fixation and enhancement: To better preserve and display the structure of biomacromolecules, secondary fixation was performed using a 1% osmium tetroxide (OsO4) aqueous solution at room temperature in the dark for 1 hour; after fixation, the cells were thoroughly rinsed with ultrapure water.
[0049] c. Dehydration: The sample was dehydrated by passing it through a series of gradient ethanol solutions: 50%, 70%, 80%, and 90% ethanol once each, for 15 minutes each time; finally, it was treated with 100% anhydrous ethanol three times, for 20 minutes each time, to ensure that the water was completely replaced.
[0050] d. Drying: To avoid damage to the ultrastructure caused by surface tension, the critical point drying method was used. The sample was transferred from anhydrous ethanol to an intermediate solution (isoamyl acetate) and immersed for 30 minutes. Then, it was placed in a critical point dryer, using liquid CO2 as the displacement medium, and the drying was completed under precisely controlled temperature and pressure.
[0051] e. Gold sputtering: The dried film sample is fixed to the metal sample stage with conductive double-sided tape and placed in an ion sputtering instrument. Under vacuum conditions, a gold-palladium alloy with a thickness of about 10 nm is sputtered onto the sample surface to enhance the conductivity and secondary electron emission rate of the sample.
[0052] 2. SEM observation and image acquisition: a. Place the prepared sample stage into the sample chamber of the scanning electron microscope.
[0053] b. Operate in high vacuum mode and set the accelerating voltage to 5-10 kV to reduce electron beam damage to the sample while obtaining good image contrast.
[0054] c. Observe the surface morphology of the membrane at different magnifications.
[0055] 3. Results: In Example 1, the cells were tightly packed and interconnected, forming a continuous and dense cell layer. The cells had a strong three-dimensional appearance, were plump, and had clearly distinguishable cell boundaries, exhibiting vigorous growth and good cytoskeleton development. This group of cells showed extremely high activity and tight intercellular connections. This robust cell layer structure is the basis for forming a tough and intact cell membrane. The addition of NAD+ precursor (NMN) not only promoted the secretion of extracellular matrix, but also significantly enhanced the vitality, spreading ability, and cell-cell interactions of amniotic stem cells. The resulting tight and three-dimensional cell layer structure is one of the fundamental guarantees for the excellent mechanical integrity and functionality of the membrane, which is completely consistent with the observed macroscopic properties of the membrane, such as ease of manipulation and good toughness.
[0056] Example 6 Optimized analysis of the elastic modulus of amniotic stem cell membranes, such as Figure 5 As shown.
[0057] It should be noted that, for the sake of simplicity, the foregoing embodiments are all described as a series of actions. However, those skilled in the art should understand that the present invention is not limited to the described order of actions, as some steps may be performed in other orders or simultaneously according to the present invention. Furthermore, those skilled in the art should also understand that the embodiments described in the specification are preferred embodiments, and the actions and modules involved are not necessarily essential to the present invention.
[0058] It should be understood that the disclosed apparatus can be implemented in other ways, as illustrated in the embodiments provided in this application. For example, the apparatus embodiments described above are merely illustrative; the division of units described above is only a logical functional division, and in actual implementation, there may be other division methods. For example, multiple units or components may be combined or integrated into another system, or some features may be ignored or not executed. Furthermore, the coupling or communication connections shown or discussed may be through some interfaces; the indirect coupling or communication connections between devices or units may be telecommunications or other forms.
[0059] The units described above as separate components may or may not be physically separate. The components shown as units may or may not be physical units; that is, they may be located in one place or distributed across multiple network units. Some or all of the units can be selected to achieve the purpose of this embodiment according to actual needs.
[0060] The above embodiments are only used to illustrate the technical solutions of the present invention, and are not intended to limit the scope of protection of the invention. Obviously, the described embodiments are only some embodiments of the present invention, not all embodiments. Based on these embodiments, all other embodiments obtained by those skilled in the art without creative effort are within the scope of protection of the present invention. Although the present invention has been described in detail with reference to the above embodiments, those skilled in the art can still combine, add, delete, or otherwise adjust the features of the various embodiments of the present invention according to the circumstances without conflict or creative effort, thereby obtaining different technical solutions that do not fundamentally depart from the concept of the present invention. These technical solutions also fall within the scope of protection of the present invention.
Claims
1. An optimized method for preparing amniotic stem cell membrane sheets, characterized in that, Includes the following steps: (1) Amniotic stem cells were seeded into a culture dish and cultured. (2) When the cells adhere to the wall and grow to a degree of confluence of more than 80%, replace the medium with an induction medium containing vitamin C; during the second medium change, add NAD+ precursor compounds to the induction medium containing vitamin C. (3) Continue culturing for 7-10 days, changing the induction medium regularly during this period, until a complete cell membrane is formed; (4) The cell membrane sheet is mechanically peeled off from the bottom of the culture dish to obtain an intact amniotic stem cell membrane sheet.
2. The optimized method for preparing amniotic stem cell membranes according to claim 1, characterized in that, The concentration of vitamin C in the induction medium is 10-100 μg / mL.
3. The optimized method for preparing amniotic stem cell membranes according to claim 2, characterized in that, The concentration of vitamin C in the induction medium was 20 μg / mL.
4. The optimized method for preparing amniotic stem cell membranes according to claim 1, characterized in that, The NAD+ precursor compound is β-nicotinamide mononucleotide (NMN) or nicotinamide adenine dinucleotide (NAD+).
5. The optimized method for preparing amniotic stem cell membranes according to claim 4, characterized in that, The induction medium contains NMN at a concentration of 0.01-0.1 mM and NAD+ at a concentration of 0.1-1 mM.
6. The optimized method for preparing amniotic stem cell membranes according to claim 5, characterized in that, The induction medium contained 0.1 mM NMN and 1 mM NAD+.
7. The optimized method for preparing amniotic stem cell membranes according to claim 1, characterized in that, The induction medium was changed every two days.
8. The optimized method for preparing amniotic stem cell membranes according to claim 1, characterized in that, In step (4), the mechanical dissection is performed using a sterile cell scraper or tweezers.
9. An amniotic stem cell membrane, characterized in that, Prepared using the method described in any one of claims 1-8, the membrane contains less than 0.12% CD34-positive cells and has an intact cell-matrix structure.
10. The use of the amniotic stem cell membrane as described in claim 9 in the preparation of drugs or medical devices for treating corneal injuries, skin trauma, knee joint filling, or endometrial adhesions.